A Salmonella Paratyphi A with an O-Antigen Having an Extended Carbohydrate Chain and Use Thereof

ABSTRACT

The present invention discloses a  Salmonella Paratyphi  A with an O-antigen having an extended carbohydrate chain and uses thereof. The method comprises the following steps: inactivating an cld gene encoding an enzyme controlling chain length of O-antigen of a  Salmonella paratyphi  A strain to obtain a  Salmonella paratyphi  A with deletion of cld gene; allowing overexpression of cld LT2  gene encoding an enzyme controlling chain length of O-antigen of  Salmonella typhimurium  in  Salmonella paratyphi  A deficient in the cld gene encoding an enzyme controlling chain length of O-antigen, so as to extend carbohydrate chain length of O-antigen. Both of the  Salmonella paratyphi  A O-polysaccharide-recombinant fusion protein conjugate vaccines rCTB4573 3 -OPS Spty50973  and rEPA4573-OPS Spty50973  as prepared by using  Salmonella Paratyphi  A with an O-antigen having an extended carbohydrate chain can induce mice to generate specific antibodies against  Salmonella paratyphi  A, and their antibody titers are significantly improved.

TECHNICAL FIELD

The present invention relates to the field of biotechnology, and inparticular, relates to a Salmonella Paratyphi A with an O-Antigen havingan extended carbohydrate chain and use thereof.

BACKGROUND ART

Salmonella spp. is a highly contagious gram-negative intestinal pathogenwith strong endotoxin and invasiveness, belongs to intracellularbacteria, and can attach small intestinal mucosal to cause diseases suchas enteric fever, gastroenteritis and septicemia, even severe intestinalbleeding or perforation. For most serotypes of Salmonella, their medianinfective doses are between 10⁵ and 10⁸, but in epidemic outbreaks, theinfective doses are generally less than 10³ bacteria, sometimes evenless than 100 bacteria. In Asian countries, especially in China, theproportion of intestinal diseases caused by Salmonella paratyphi A isincreasing, and some studies find that there are 150 cases of Salmonellaparatyphi A infection per 100 000 people per year.

At present, the main way to treat typhoid and paratyphoid isantibiotics, but with the emergence of drug-resistance, especially theemergence of multiple drug-resistant strains, conventional antibiotictreatment encounters a huge challenge, and immunization of relevantvaccines is an effective means of prevention. At present, the progressesof research and development for oral attenuated live vaccine againstSalmonella typhimurium, Vi capsular polysaccharide vaccine and Vipolysaccharide-protein conjugate vaccine are rapid, and there are avariety of products listed, but these vaccines are not able to generatecross immunoprotection against Salmonella paratyphi A. Currently, thereis not a vaccine against Salmonella paratyphi A that has been approvedfor listing.

CONTENTS OF THE INVENTION

One object of the present invention is to provide a recombinant strain.

The recombinant strain as provided by the present invention is obtainedby introducing cld_(LT2) gene encoding an enzyme controlling chainlength of O-antigen of Salmonella typhimurium into Salmonella paratyphiA deficient in cld gene encoding an enzyme controlling chain length ofO-antigen.

In the above recombinant strain, the enzyme controlling chain length ofO-antigen of Salmonella typhimurium has an amino acid sequence with atleast 90% identity to the amino acid sequence as shown in SEQ ID NO: 2;

the enzyme (also named as Cld_(LT2)) controlling chain length ofO-antigen of Salmonella typhimurium has an amino acid sequence as shownin SEQ ID NO: 2;

the enzyme Cld_(LT2) controlling chain length of O-antigen of Salmonellatyphimurium has a coding sequence as shown in SEQ ID NO: 1.

In the above recombinant strain, Salmonella paratyphi A deficient in cldgene encoding an enzyme controlling chain length of O-antigen isobtained by knocking out cld gene via Red recombination technology, orby knocking out cld gene via homologous recombination, or by insertingan inactivated cld gene, or the cld deficient strain can be obtained byinduced mutation; preferably, is obtained by knocking out cld gene viaRed recombination technology.

In the above recombinant strain, Salmonella paratyphi A deficient in cldgene encoding an enzyme controlling chain length of O-antigen isobtained by a method comprising the following steps:

(1) preparing a linear targeting DNA fragment 1, which has a nucleotidesequence as shown in SEQ ID NO: 11, which contains a cat gene;

(2) transforming pKD46 plasmid into Salmonella. paratyphi, to obtain arecombinant strain designated as S. paratyphi/pKD46;

(3) inducing expression of Red recombination system in the S.paratyphi/pKD46 strain, and transforming the linear targeting DNAfragment 1 into the S. paratyphi/pKD46 strain, so that the lineartargeting DNA fragment 1 replaces the cld gene in the S. paratyphi/pKD46strain to obtain a recombinant strain designated as S. paratyphicld::cat/pKD46;

(4) deleting the pKD46 plasmid from the S. paratyphi cld::cat/pKD46strain, to obtain a recombinant strain designated as S. paratyphicld::cat;

the S. paratyphi cld::cat is a S. paratyphi in which cld gene sequenceis substituted with cat gene sequence;

(5) transforming plasmid pCP20 into the S. paratyphi cld::cat anddeleting cat gene, to obtain a recombinant strain designated as S.paratyphi Δcld;

the S. paratyphi Acid is a cld gene-deleted S. paratyphi.

Another object of the present invention is to provide a method forextending carbohydrate chain length of O-antigen of Salmonella paratyphiA.

The method for extending carbohydrate chain length of O-antigen ofSalmonella paratyphi A as provided in the present invention comprisesthe following steps: culturing the above recombinant strain to expressthe cld_(LT2) gene, so that the recombinant strain synthesizes anO-antigen of which carbohydrate chain length is extended.

It is also an object of the present invention to provide an O-antigen.

The O-antigen provided by the present invention is prepared according tothe method described above.

The use of the above-described O-antigen in the manufacture of a productfor the prevention or prophylaxis of diseases caused by Salmonellaparatyphi A is also within the scope of the present invention.

It is a further object of the present invention to provide an method forpreparing a vaccine for the prevention and/or treatment of diseasescaused by Salmonella paratyphi A via one-step bio-crosslinking.

The for preparing a vaccine for the prevention and/or treatment ofdiseases caused by Salmonella paratyphi A as provided in the presentinvention via one-step bio-crosslinking method comprises the steps of:

(1) inactivating cld gene encoding an enzyme controlling chain length ofO-antigen of Salmonella paratyphi A and waaL gene encoding O-antigenligase of Salmonella paratyphi A, to obtain a Salmonella paratyphi Awith double deletion of cld gene and waaL gene;

(2) introducing a cld_(LT2) gene encoding an enzyme controlling chainlength of O-antigen of Salmonella typhimurium, a pglL gene encodingO-oligosaccharyltransferase of Neisseria meningitidis and a geneencoding recombinant fusion protein into the Salmonella paratyphi A withdouble deletion of cld gene and waaL gene, to obtain a recombinantstrain;

(3) culturing the recombinant strain to obtain recombinant fusionprotein with O-antigen-modified, and processing the recombinant fusionprotein with O-antigen-modified to obtain the target vaccine.

In the above method, the Salmonella paratyphi A with double deletion ofcld gene encoding an enzyme controlling chain length of O-antigen andwaaL gene encoding O-antigen ligase can be obtain by knocking out cldgene and waaL gene via Red recombination technology, or by knocking outcld gene and waaL gene via homologous recombination, or by inserting aninactivated cld gene and waaL gene, or by obtaining a stain withdeletion of cld and waaL genes by induced mutation; preferably, isobtained via Red recombination technology.

In the above method, the Salmonella paratyphi A with double deletion ofcld gene encoding an enzyme controlling chain length of O-antigen andwaaL gene encoding O-antigen ligase is constructed by a methodcomprising the following steps:

(1) preparing a linear targeting DNA fragment 2, which has a nucleotidesequence as shown in SEQ ID NO: 12, which contains a kan gene;

(2) transforming pKOBEG plasmid into the S. ParatyphiΔcld strain, toobtain a recombinant strain designated as S. paratyphiΔcld/pKOBEG;

(3) inducing expression of Red recombination system in the S.paratyphiΔcld/pKOBEG strain, and transforming the linear targeting DNAfragment 2 into the S. paratyphiΔcld/pKOBEG strain, so that the lineartargeting DNA fragment 2 replaces the waaL gene, in the S.paratyphiΔcld/pKOBEG strain to obtain a recombinant strain designated asS. ParatyphiΔcld waal::kan/pKOBEG;

(4) deleting the pKOBEG plasmid from the S. paratyphiΔcldwaal::kan/pKOBEG strain, to obtain a recombinant strain designated as S.paratyphiΔcldwaaL::kan;

the S. paratyphiΔcldwaaL::kan is a S. paratyphiΔcld in which waaL genesequence is substituted with kan gene sequence;

(5) transforming plasmid pCP20 into the S. paratyphiΔcldwaaL::kan anddeleting kan gene, to obtain a recombinant strain designated as S.paratyphiΔcldΔwaaL;

the S. paratyphiΔcldΔwaaL is a S. paratyphi with deletion of cld geneand waaL gene.

In the above method, the recombinant fusion protein comprises aN-terminal signal peptide, a carrier protein, and a peptide fragmentcomprising a serine as an O-glycosylation site at position 63 ofNeisseria meningitidis pilin PilE.

In the above method, the carrier protein is a non-toxic mutant of abacterial toxin protein or a fragment of a bacterial toxin protein.

In the above method, the bacterial toxin protein is Pseudomonasaeruginosa exotoxin A, cholera toxin, diphtheria toxin or tetanus toxin.

In the above method, the non-toxic mutant of the bacterial toxin proteinis a non-toxic mutant of Pseudomonas aeruginosa exotoxin A or anon-toxic mutant of diphtheria toxin; the fragment of the bacterialtoxin protein is a B subunit of cholera toxin or a C protein of tetanustoxin.

In the above method, the carrier protein is specifically a non-toxicmutant of Pseudomonas aeruginosa exotoxin A or a B subunit of choleratoxin.

In the above method, the peptide fragment comprising a serine as anO-glycosylation site at position 63 of Neisseria meningitidis pilin PilEis a peptide fragment having an amino acid sequence as set forth inpositions 45-73 of Neisseria meningitidis pilin PilE.

In the above method, the N-terminal signal peptide can be a signalpeptide such as PelB, DsbA, STII, OmpA, PhoA, LamB, SpA, Enx, and theN-terminal signal peptide is specifically a DsbA signal peptide.

In the above method, the cld_(LT2) gene encoding an enzyme controllingchain length of O-antigen of Salmonella typhimurium, the pglL geneencoding O-oligosaccharide transferase of Neisseria meningitidis and agene encoding the recombinant fusion protein can be separatelyconstructed into different recombinant expression vectors and introducedinto the Salmonella paratyphi A with double deletion of cld and waaL, orcan be constructed into the same recombinant expression vector andintroduced into the Salmonella paratyphi A with double deletion of cldand waaL, or can also be introduced into the Salmonella paratyphi A withdouble deletion of cld and waaL via separately incorporating them into ahost genome; the cld_(LT2) gene encoding an enzyme controlling chainlength of O-antigen of Salmonella typhimurium, the pglL gene encodingO-oligosaccharide transferase of Neisseria meningitidis and the geneencoding the recombinant fusion protein can be controlled by aninducible promoter, or controlled by a constitutive promoter.

In the above method, the cld_(LT2) gene encoding an enzyme controllingchain length of O-antigen of Salmonella typhimurium, the pglL geneencoding O-oligosaccharide transferase of Neisseria meningitidis arespecifically introduced into the Salmonella paratyphi A with doubledeletion of cld gene encoding an enzyme controlling chain length ofO-antigen and waaL gene encoding O-antigen ligase viapETtac28-pglL-cld_(LT2) recombinant expression vector.

In the above method, the gene encoding the recombinant fusion protein isspecifically introduced into the Salmonella paratyphi A with doubledeletion of cld gene encoding an enzyme controlling chain length ofO-antigen and waaL gene encoding O-antigen ligase via pMMB66EH-rCTB4573recombinant expression vector or pMMB66EH-rEPA4573 recombinantexpression vector or pMMB66EH-rCTB4573₃ recombinant expression vector.

In the above method, the pETtac28-pglL-cld_(LT2) recombinant expressionvector is constructed by a method comprising: using restrictionendonucleases EcoR I and Hind III to perform double digestion ofcld_(LT2) gene encoding an enzyme controlling chain length of O-antigenof Salmonella typhimurium and the pMMB66EH vector, ligating to obtain atransition vector pMMB66EH-cld_(LT2); using the transition vectorpMMB66EH-cld_(LT2) as template, amplifying to obtain an expressioncassette of cld_(LT2), using restriction endonuclease Xba I and Xho I toperform double digestion of the expression cassette of cld_(LT2) andpET28a vector, ligating to obtain pETtac28-cld_(LT2) vector; usingrestriction endonucleases EcoR I and Hind III to perform doubledigestion of O-oligosaccharide transferase gene pglL of Neisseriameningitidis and pKK223-3 vector, ligating to obtain pKK223-3-pglLvector; using pKK223-3-pglL vector as template, amplifying to obtain anexpression cassette of PglL; using restriction endonuclease Bgl II toperform double digestion of the expression cassette of PglL and thepETtac28-cld_(LT2) vector, ligating to obtain thepETtac28-pglL-cld_(LT2) recombinant expression vector.

In the above method, the pMMB66EH-rEPA4573 recombinant expression vectoris constructed by a method comprising: ligating the gene encoding fusionprotein rEPA4573 of recombinant Pseudomonas aeruginosa exotoxin A into amultiple cloning site of pMMB66EH vector to obtain the pMMB66EH-rEPA4573recombinant expression vector.

In the above method, the pMMB66EH-rCTB4573 recombinant expression vectoris constructed by a method comprising: ligating the gene encoding fusionprotein rCTB4573 of recombinant cholera toxin B subunit into a multiplecloning site of pMMB66EH vector to obtain the pMMB66EH-rCTB4573recombinant expression vector.

In the above method, the pMMB66EH-rCTB4573₃ recombinant expressionvector is constructed by a method comprising: ligating the gene encodingfusion protein rCTB4573₃ of recombinant cholera toxin B subunit into amultiple cloning site of pMMB66EH vector to obtain thepMMB66EH-rCTB4573₃ recombinant expression vector.

In the above method, the enzyme Cld_(LT2) controlling chain length ofO-antigen of Salmonella typhimurium has an amino acid sequence as shownin SEQ ID NO: 2; and gene encoding the enzyme controlling chain lengthof O-antigen of Salmonella typhimurium has a sequence as shown in SEQ IDNO: 1;

the Neisseria meningitides oligosaccharyltransferase pglL has an aminoacid sequence as shown in SEQ ID NO: 8; the gene encoding Neisseriameningitides oligosaccharyltransferase pglL has a sequence as shown inSEQ ID NO: 7;

the fusion protein rEPA4573 of recombinant Pseudomonas aeruginosaexotoxin A has an amino acid sequence as shown in SEQ ID NO: 4; the Nos1-19 from the N-terminal of the sequence shown in SEQ ID NO: 4 are of anamino acid sequence of the DsbA signal peptide; the amino acids at Nos20-631 are of an amino acid sequence of the non-toxic mutant ofPseudomonas aeruginosa exotoxin A; the Nos 632-636 are of a flexiblelinker sequence; the amino acids at Nos 637-665 are of an amino acidsequence of a peptide at sites 45-73 of Neisseria meningitidi spilinPilE (NC_003112.2); the Nos 666-674 are of a flexible linker sequenceand a 6×His tag sequence; the fusion protein rEPA4573 of recombinantPseudomonas aeruginosa exotoxin A has a coding sequence as shown in SEQID NO: 3;

the fusion protein rCTB4573 of recombinant cholera toxin B subunit hasan amino acid sequence as shown in SEQ ID NO: 6; the Nos 1-19 from theN-terminal of the sequence shown in SEQ ID NO: 6 are of an amino acidsequence of the DsbA signal peptide; the Nos 20-122 are of amino acidsequence of cholera toxin B subunit; the Nos 123-127 are of a flexiblelinker sequence; the Nos 128-156 are of an amino acid sequence of apeptide at sites 45-73 of Neisseria meningitidis pilin PilE(NC_003112.2); the Nos 157-166 are of a flexible linker and a 6×His tagsequence; the fusion protein rCTB4573 of recombinant cholera toxin Bsubunit has a coding sequence as shown in SEQ ID NO: 5;

the fusion protein rCTB4573₃ of recombinant cholera toxin B subunit hasan amino acid sequence as shown in SEQ ID NO: 10; the Nos 1-19 from theN-terminal of the sequence shown in SEQ ID NO: 10 are of an amino acidsequence of the DsbA signal peptide; the Nos 20-122 are of amino acidsequence of cholera toxin B subunit; the Nos 123-127 are of a flexiblelinker sequence; the Nos 128-222 are of an amino acid sequence of 3repetitive peptides of that at sites 45-73 of Neisseria meningitidispilin PilE (NC_003112.2); the Nos 223-232 are of a flexible linker and a6×His tag sequence; the fusion protein rCTB4573₃ of recombinant choleratoxin B subunit has a coding sequence as shown in SEQ ID NO: 9;

the peptide as set forth in amino acid position 45-73 of Neisseriameningitidis pilin PilE has an amino acid sequence as shown in SEQ IDNO: 14; and gene encoding the peptide has a sequence as shown in SEQ IDNO: 13.

The vaccines as prepared according to the above methods also fall intothe protection scope of the present invention.

The uses of the vaccines in manufacture of a product for prophylaxisand/or treatment of a disease caused by Salmonella paratyphi A also fallinto the protection scope of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a PCR identification diagram of S. paratyphi CMCC50973 withknockout of cld gene.

FIG. 2 shows a silver staining diagram of LPS before and after S.paratyphi CMCC50973 cld gene is replaced.

FIG. 3 shows a detection diagram of glycosylation modification of therecombinant fusion protein after the substitution of cld gene.

FIG. 4 shows a detection diagram of multi-site glycosylationmodification of the recombinant CTB fusion proteins.

FIG. 5 shows a detection diagram of rEPA4573-OPS_(Spty50973)purification effect.

FIG. 6 shows a detection diagram of rCTB4573₃-OPS_(Sp50973) purificationeffect.

FIG. 7 shows an immunogenicity evaluation of glycoprotein conjugates.

FIG. 8 shows a molecular identification of S. paratyphiCMCC50973ΔcldΔwaaL.

BEST MODELS FOR CARRYING OUT THE INVENTION

The experimental methods used in the following examples are conventionalmethods unless otherwise specified.

The materials, reagents and the like used in the following examples arecommercially available, unless otherwise specified.

Experimental materials: Salmonella paratyphi A CMCC50973 strain(Salmonella. paratyphi CMCC50973), purchased from the China MedicalBacteria Deposit Management Center.

pET-22b plasmid, purchased from Novagen Corporation under catalog number69744.

pKOBEG plasmid, disclosed in the literature “A rapid method forefficient gene replacement in the filamentous fungus Aspergillusnidulans [J]. Nucleic Acids Res. 2000 Nov. 15; 28 (22): E97”, availablefor the public in the Institute of Bioengineering of the Academy ofMilitary Medical Sciences of the Chinese People's Liberation Army; theplasmid is a temperature-sensitive plasmid with chloramphenicolresistance.

pKD3 plasmid, disclosed in the literature “Datsenko, K. A. and B. L.Wanner, One-step inactivation of chromosomal genes in Escherichia coliK-12 using PCR products. Proc Natl Acad Sci USA, 2000, 97(12): p.6640-5.”; available for the public in the Institute of Bioengineering ofthe Academy of Military Medical Sciences of the Chinese People'sLiberation Army.

pKD46 plasmid, discloses in the literature “Datsenko, K. A. and B. L.Warmer, One-step inactivation of chromosomal genes in Escherichia coliK-12 using PCR products. Proc Natl Acad Sci USA, 2000, 97(12): p.6640-5.”; available for the public in the Institute of Bioengineering ofthe Academy of Military Medical Sciences of the Chinese People'sLiberation Army. The replicons of the pKD46 plasmid are temperaturesensitive, which would be disappeared during culture at 37° C., and thisplasmid contains DNA encoding three recombinases for the Redrecombination system, which is controlled by arabinose promoter.

pCP20 plasmid, disclosed in the literature “Datsenko, K. A. and B. L.Wanner, One-step inactivation of chromosomal genes in Escherichia coliK-12 using PCR products. Proc Natl Acad Sci USA, 2000, 97(12): p.6640-5.”; available for the public in the Institute of Bioengineering ofthe Academy of Military Medical Sciences of the Chinese People'sLiberation Army. The replicons of the pCP20 plasmid are temperaturesensitive, which be lost during culture at 42° C. The plasmid containsDNA encoding FLP recombinase, which is controlled by a thermo-sensitivepromoter, and the expression of FLP recombinase is induced at 42° C.,while the plasmid is lost.

Aluminum hydroxide adjuvant Rehydragel LV was purchased from GeneralChemical Company.

Example 1. Method for Extending Length of Salmonella paratyphi AO-Antigen Carbohydrate Chain

I. Knocking Out cld Gene Encoding an Enzyme Controlling Chain Length ofO-Antigen of Salmonella paratyphi A CMCC50973 Strain

1. Preparation of Linear Targeting DNA Fragment 1

1) Design of PCR Primer

The targeting fragment of the cld gene (sites 887812 to 888789) wasdesigned according to the S. paratyphi ATCC9150 genome sequence(CP000026) published by GeneBank, and the cld gene of Salmonellaparatyphi A CMCC50973 was knocked out. In the upstream and downstream ofthe cld gene, 500 bp fragments were cut out and used as the homologousarms, and the targeting fragment of chloramphenicol resistant genecontaining 500 bp homologous sequence and FRT site at both ends wasamplified by PCR. 50973cld up 5′ and 50973cld down 3′ were used toidentify whether the cld gene was knocked out. The specific primerdesign was shown in Table 1.

TABLE 1  Primers used for knocking out cld gene ofSalmonella paratyphi A CMCC50973 strain Effect Name of Primer sequence of primer primer (5′→3′) Notes pair 50973 cld CCAGCTTCATCCTTTTTTTAUnder- Amplifying cat 5′ GTTAGGGTATCTATGACAAG lined to obtainCGATTGTGTAGGCTGGAG part chloram- 50973 cld CCTTTCGAAGCCGACCACCA was cldphenicol cat 3′ TCCGGCAAAGAAGCTAATTA homo- resistantACGGCTGACATGGGAATTAG logous gene arm. fragments with cld upstream anddownstream homologous arms. 50973 cld CAGTTGCTGGCTAATTATCA — Amplifyingup 5′ GTCAGTGCCT cld 50973 cld GTCATAGATACCCTAACTAA primers of up 3′AAAAAGGATGAAGC upstream 50973 cld TTCTTTGCCGGATGGTGGTC and down 5′GGCTTCGAAA downstream 50973 cld TTATGGACCAAAGGCGAAAC homologous down 3′CTCAGGCCAT arms.

2) Acquisition of Linear Targeting DNA Fragment 1

Using the plasmid pKD3 as template, primers 50973 cld cat 5′ and 50973cld cat 3′ were used to amplify a chloramphenicol resistant genefragment 50973 cld cat which had at both ends homologous arms 41 bpupstream and downstream of the cld gene and FRT site; using Salmonellaparatyphi A CMCC50973 genomic DNA as template, amplification wasperformed by using 50973 cld up5′ and 50973 cld up3′ to obtain cldupstream 500 bp homologous arm 50973 cld up, while using 50973 cld down5′ and 50973 cld down 3′ to obtained cld downstream 500 bp homologousarm 50973 cld down, and then the 3 fragments 50973 cld cat, 50973 cld upand 50973 cld down were fused by PCR to obtain linear targeting DNAfragment 1 (SEQ ID NO: 11).

PCR reaction system was of 50 μL, which comprised Q5 super-fidelity DNApolymerase 0.5 μL, 5× buffer 10 μL, template 1 μL, dNTP 4 μL, primer 2.5μL, deionized water 29.411;

PCR reaction conditions: 98° C. 20 s; 98° C. 10 s, 55° C. 10 s, 72° C.50 s, 30 cycles; 72° C. 5 min, to obtain the linear targeting DNAfragment 1 as shown in SEQ ID NO: 11. The product was separated by 1%agarose gel electrophoresis, the target strip was cut out from the gel,and the PCR product was recovered by using a DNA gel recovery kit. Theprocedure was carried out according to the instructions.

2. Acquisition of Salmonella paratyphi A (S. paratyphi CMCC50973Δcld)Deficient in cld Gene Encoding an Enzyme Controlling Chain Length ofO-Antigen

1) Preparation of S. paratyphi CMCC50973/pKD46

(1) S. paratyphi CMCC50973 was inoculated in a LB liquid medium,incubated overnight at 37° C., transferred to LB liquid medium at 1:100,and incubated at 37° C. until OD₆₀₀ reached 0.6.

(2) Bacteria were collected by centrifugation, washed with autoclavationsterilized 10% glycerol (v/v) for four times, and finally were suspendedwith 400 μL of 10% glycerol, to obtain competent cells forelectroporation, which were sub-packaged, ready to use.

(3) pKD46 plasmid was electroporated into the prepared S. paratyphiCMCC50973 competent cells, coated on a LB plates containing 100 μg/mLampicillin, cultured at 30° C. overnight, to obtain positive clones asS. paratyphi CMCC50973/pKD46 strain.

2) Acquisition of S. paratyphi CMCC50973 cld::cat Strain

(1) The above obtained S. paratyphi CMCC50973/pKD46 was culturedovernight at 30° C. and passed at 1:100 in a LB liquid medium containingampicillin.

(2) When OD₆₀₀ was about 0.2, L-arabinose at a final concentration of0.2% (w/v) was added to induce the expression of Red recombinationsystem, and when OD₆₀₀ value was 0.6, S. paratyphi CMCC50973/pKD46competent cells were prepared by the steps as described above.

(3) 10 μL of the linear targeting DNA fragment 1 obtained in the aboveStep 1 was taken and electroporated into the sub-packaged S. paratyphiCMCC50973/pKD46 competent cells.

(4) The cells were rapidly added to 1 mL of pre-cooled LB medium,resuscitated at 30° C. for about 2.5 h, and then coated on a LB platecontaining 30 μg/mL chloramphenicol at a final concentration, and placedin a 30° C. incubator for culture overnight.

(5) The positive clones were screened, and subjected to PCRidentification using primers 50973 cld cat 5′ and 50973 cld cat 3′, inwhich the wild-type bacteria could not be amplified to generatefragments, the cld gene was identified to have a size of 1100 bp afterbeing substituted by cat gene. According to the PCR results, positiveclones of S. Paratyphi CMCC50973 cells in which the cld gene wasreplaced by the cat gene were screened out, to obtain the deletionmutant S. paratyphi CMCC50973 cld::cat/pKD46.

3) Acquisition of S. paratyphi CMCC50973Δcld Strain

(1) The positive clone S. paratyphi CMCC50973 cld::cat/pKD46 wasinoculated into a LB liquid medium containing 30 μg/mL chloramphenicol,cultured and passed at 37° C. for three times (cultured for 12 h eachtime), to remove pKD46 plasmid, so as to obtain S. paratyphi CMCC50973cld::cat strain.

(2) The S. paratyphi CMCC50973 cld::cat as prepared according to theabove method was used for electroporation of the competent cells, andplasmid pCP20 was electroporated into S. paratyphi CMCC50973 cld::catcompetent cells, and then coated with a LB plate containing ampicillin,so as to obtain S. paratyphi CMCC50973 cld::cat/pCP20 strain.

(3) S. paratyphi CMCC50973 cld::cat/pCP20 monoclones were picked andplaced in a LB liquid medium, incubated at 30° C. until OD₆₀₀ was about0.6, and transferred and cultured at 42° C. overnight.

(4) The bacterial solution cultured overnight at 42° C. was streaked ona LB plate, and monoclones were picked out on LB plates and LB platescontaining chloramphenicol. The monoclones that grew on the LB platesbut did not grow on chloramphenicol-containing LB plates were chosen,and subjected to PCR identification by using primers 50973 cld up 5′ and50973 cld down 3′. The PCR identification showed the size of wild S.paratyphi CMCC50973 fragment was 2028 bp, while the size after knockoutof the cld gene was 1100 bp. The results of the PCR identification wereshown in FIG. 1. The positive clone was named S. paratyphiCMCC50973Δcld.

II. Acquisition and Phenotype Identification of O-Antigen CarbohydrateChain-Extended Salmonella paratyphi A

1. Construction of a Vector Expressing cld_(LT2) Gene Encoding an EnzymeControlling Chain Length of O-Antigen of Salmonella typhimurium

The DNA sequence as shown in SEQ ID NO: 1 was synthesized according tothe nucleotide sequence of cld gene (GeneBank No.: NC_003197.1, position2156832 to 2157815) encoding an enzyme controlling chain length ofO-antigen of Salmonella typhimurium, and this gene was named ascld_(LT2). The enzyme controlling chain length of O-antigen has an aminoacid sequence as shown in SEQ ID NO: 2, and the cld_(LT2) gene encodingthe enzyme has a sequence as shown in SEQ ID NO: 1.

The synthesized cld_(LT2) gene was digested with EcoRI and Hind III,linked into pMMB66EH expression vector (purchased from ATCC, ATCC37620),to construct transition vector pMMB66EH-cld_(LT2), the primers 66tac 5′and 66tac 3′ were designed by using pMMB66EH-cld_(LT2) as template, theexpression cassette carrying cld_(LT2) gene was amplified, and linked topET28a (purchased from Novagen) between restriction enzyme cutting sitesof Xba I and XhoI, so as to construct expression vectorpETtac28-cld_(LT2). The primers were as follows:

66tac 5′: AAAATCTAGAGCGCCGACATCATAACGGTTCTGGCA;66tac 3′: TTTTCTCGAGCGTTCACCGACAAACAACAGATAA.

The sequencing results showed that the sequence as shown in SEQ ID NO: 1was inserted between the restriction enzyme cutting sites of Xba I andXho I in the pET28a vector, which indicated the vector was correct.

2. Salmonella paratyphi A LPS Length Detection

The above-prepared S. paratyphi CMCC50973Δcld electroporation competentcells were electroporated with the above-obtained pETtac28-cld_(LT2),coated on kanamycin-containing LB plates, and positive clones werepicked and cultured at 37° C. When OD₆₀₀ was about 0.6, IPTG with afinal concentration of 1 mM was added to induce expression of cld_(LT2)gene.

After 10 h of induction, 1 mL of culture was taken and the cells werecollected by centrifugation, washed with PBS once, and 100 μL of lysisbuffer (2 mL of 10% SDS, 0.4 mL of 2-mercaptoethanol, 1 mL of 100%glycerol, 5 mL of 2M Tris-HCL pH 6.8, 20 μL of 10% bromophenol blue, 1.6mL of ddH₂O) was added and fully mixed, heated with boiling water for 10min, added with 4 μL of 20 mg/mL of proteinase K, reacted at 60° C. for1-2 h, 15 μL of product was taken for loading and performing SDS-PAGE,in which the separation gel was 15%, the concentrated gel was 4%, andthe electrophoresis was ceased after the bromophenol blue gel exuded for30 minutes.

The above-mentioned polyacrylamide gel was placed in an immobilizedsolution overnight, added with a sensitizing solution and reacted for 30min, washed with deionized water 3 times, 15 min per time, added asilver nitrate solution and reacted for 20 min, washed twice withdeionized water, 1 min per time, add with a developer solution for colordevelopment, the reaction was finally terminated, and the product waswashed with deionized water. At the same time, the wild strain of S.paratyphi CMCC50973 was used as control.

The results were showed in FIG. 2, in which the result of silverstaining test showed that the LPS of the wild S. paratyphi CMCC50973 wasdistributed in the lower molecular weight region, while when the cldgene thereof was replaced by cld_(LT2), relatively concentrated LPSstripes appeared in the higher molecular weight region, which indicatedthat by replacing the cld gene of S. paratyphi CMCC50973 with cld_(LT2),the O-antigen carbohydrate chain thereof was significantly extended.

Example 2. Salmonella paratyphi A O-Antigen-Recombinant Fusion ProteinConjugate Vaccine as Prepared by One-Step Bio-Crosslinking Method andUses Thereof

I. Construction of O-Antigen Ligase Gene waaL Deficient Salmonellaparatyphi A

(I) Preparation of Linear Targeting DNA Fragment 2

1. Design and Synthesis of PCR Primers

By referring to the waaL gene (GeneBank No. CP000026, sites3696236-3697450) in the whole genome sequence (NC_006511) of theSalmonella paratyphi A 50973 strain (S. paratyphi CMCC50973) and itsupstream and downstream sequences, a pair of primers was designed foreach of upstream (5′ end) and downstream (3′ end) of the waaL gene,namely 73waaLu1/73 waaLu2 and 73waaLd1/73waaLd2. For ease ofmanipulation, the restriction sites BamH I and Sal I were added to theprimer ends of the upstream homologous arm up, and the restriction sitesHind III and Xho I were added to the primer ends of the downstreamhomologous arm down. In the meantime, besides the waaL gene upstream anddownstream homologous arms on the genome, a pair of primers(73waaLw1/73waaLw2), a pair of internal detection primers(73waaLn1/73waaLn2) and a pair of kan gene primers (Kan1/Kan2) weredesigned for simultaneous sequencing and verification, in order to testwhether the mutants were constructed successfully. The above-mentionedprimers had sequences as shown in Table 2 (the underlined sequences wererecognition sites for restriction enzyme digestion).

TABLE 2  Sequences of primers Primer Sequence (5′→3′) Use 73waaLu1CGGGATCCAGGCTTTGACTATGTGGA Amplifying  73waaLu2GCGTCGACATCTGGCGATATGAGTATG up fragment 73waaLd1CCAAGCTTTAGTGCAGGCATATTGGG Amplifying  73waaLd2CCCTCGAGTATCACCTCGCAGAACCT down fragment 73waaLw1 AACACCGGATTACGGATAAIdentifying 73waaLw2 TGCATGGTGGCTGTAGAA mutants 73waaLn1AGAAACGGTTGCGAAAAT Identifying 73waaLn2 ATAGCCGTAGCCCTTGAT mutants Kan1GCGTCGACGTGTAGGCTGGAGCTGCTTC Identifying Kan2CCAAGCTTATGGGAATTAGCCATGGTCC mutants

2. Construction of Linear Targeting DNA Fragment 2

1) Using the genomic DNA of Salmonella paratyphi A 50973 as template,the upstream and downstream homologous arms, up fragment and downfragment, of the waaL gene were amplified by 73waaLu1/73waaLu2 and73waaLd1/73waaLd2, respectively. The PCR procedures were as follows: 94°C. 10 min; 94° C. 30 s, 50° C. 30 s, 72° C. 30 s, 30 cycles; 72° C. 10min.

2) Construction of pETKan

The kan gene fragment represented by the nucleotide sequence at sites578-2073 in SEQ ID NO: 12 was artificially synthesized, and the kan genefragment and pET-22b plasmid were digested with the restriction enzymesSalI and Hind IIII, and a recombinant plasmid was obtained afterligating, and named as pETKan. The pETKan was sequenced and the resultswere correct.

3) The up fragment was doubly digested with restriction endonucleasesBamH I and SalI to obtain a gene fragment 1; the plasmid pETKan wasdoubly digested with restriction endonucleases BamH I and Sal I toobtain a vector large fragment 1; and the gene fragment 1 was ligatedwith the vector large fragment 1 to obtain an intermediate vector 1;

the down fragment was doubly digested with restriction endonuclease HindIII and Xho I to obtain a gene fragment 2; the intermediate vector 1 wasdoubly digested with restriction endonuclease Hind III and Xho I toobtain a vector large fragment 2; and the gene fragment 2 was ligatedwith the vector large fragment 2 to obtain an intermediate vector 2.

4) The intermediate vector 2 was doubly digested with restrictionendonucleases BamH I and Xho I to obtain the desired targeting DNAfragment that had homologous arms at both sides and contained the kangene in the middle part, and the fragment has a nucleotide sequence asshown in SEQ ID NO: 12. In the SEQ ID NO: 12, as counting from the 5′end, the nucleotides at sites 7-571 were the up fragment, thenucleotides at sites 578-2073 were the kan gene, and the nucleotides atsites 2080-2543 were the down fragment.

A linear targeting DNA fragment 2 (SEQ ID NO: 12) with a concentrationof up to 300 ng/μL was obtained by further PCR amplification of thetarget fragment by using the DNA fragment shown in SEQ ID NO: 12 astemplate and 73waaLu1 and 73waaLd2 as primers.

(II) Construction of S. paratyphi CMCC50973/pKOBEG

Since the pKOBEG plasmid contained the various enzymes required toencode the λ-Red recombination system, the pKOBEG plasmid waselectroporated into S. paratyphi CMCC50973 competent cells, coated tochloramphenicol resistant (pKOBEG plasmid resistance, chloramphenicol)LB plate, and cultured at 30° C. overnight to obtain a positive clone,which was named as S. paratyphi CMCC50973/pKOBEG strain.

(III) Using Linear Targeting DNA Fragment 2 to Electroporate S.paratyphi CMCC50973/pKOBEG

1. S. paratyphi CMCC50973/pKOBEG was inoculated to a low salt LB mediumcontaining chloramphenicol in a final concentration of 30 μg/mL andcultured overnight at 30° C., then passaged to a low salt LB liquidmedium at a volume ratio of 1:100, and continuously cultured.

2. The culture medium in Step 1 was added with L-arabinose at a finalconcentration of 1 mmol/L at 1 hour before the OD₆₀₀ value reached 0.6,so as to induce the expression of the Red recombination system.

3. When the OD₆₀₀ value of the culture medium in step 2 reached 0.6, 5μL of the 300 ng/μL linear targeting DNA fragment 2 as prepared in step(I) was taken and used to electroporate and transform S. paratyphiCMCC50973/pKOBEG.

4. 1 mL of pre-cooled low salt LB liquid medium was rapidly added to thetransformed cells, resuscitation was performed at 30° C. for about 2.5hours, and then the medium was coated on LB plates containing kanamycinat a concentration of 50 μg/mL, placed in 30° C. incubator and culturedovernight, and positive clones were screened out.

5. The positive clones were inoculated into a liquid LB medium (withkanamycin resistance at a concentration of 50 μg/mL), cultured andpassaged twice at 42° C. (12 hours each time) to remove pKOBEG plasmid,and finally obtain a mutant with kanamycin resistance waaL deletion,named S. paratyphi CMCC50973 waaL::kan.

(IV) The plasmid pCP20 coding FRT site-specific recombinase waselectroporated and transferred into S. paratyphi CMCC50973waaL::kan,cultured at 30° C. on a LB plate that contained chloramphenicol at aconcentration of 50 μg/mL and was free of kanamycin, positive clones ofCm^(r)Km^(s) (chloramphenicol resistant, Kanamycin sensitive) werescreened out.

(V) The positive clones screened out in step (IV) were transferred intoa liquid LB and cultured at 42° C. for 12 h to obtain a mutant withdeletion of target genes that did not contain kanamycin and plasmidpCP20, which were named as S. paratyphi CMCC50973ΔwaaL, so thatSalmonella paratyphi A with deletion of waaL gene was obtained.

II. Construction of Salmonella paratyphi A with Deletion of waaL Geneand cld Gene

(I) Preparation of Linear Targeting DNA Fragment 2

The steps were the same of the above step (I).

(II) Construction of S. paratyphi CMCC50973Δcld/pKOBEG

Since the pKOBEG plasmid contained the various enzymes required toencode the λ-Red recombination system, the pKOBEG plasmid waselectroporated and transformed into S. paratyphi CMCC50973Δcld competentcells, and coated a LB plate with chloramphenicol resistance (pKOBEGplasmid resistance, chloramphenicol), cultured at 30° C. overnight togive a positive clone named S. paratyphi CMCC50973Δcld/pKOBEG strain.

(III) Using Linear Targeting DNA Fragment 2 to Electroporate S.paratyphi CMCC50973Δcld/pKOBEG

1. S. paratyphi CMCC50973Δcld/pKOBEG was inoculated to a low salt LBliquid culture medium containing chloramphenicol at a finalconcentration of 30 μg/mL and cultured at 30° C. overnight, and thenpassaged to a low salt LB liquid medium at a volume ratio of 1:100, andcontinuously cultured.

2. The culture medium in Step 1 was added with L-arabinose at a finalconcentration of 1 mmol/L at 1 hour before the OD₆₀₀ value reached 0.6,so as to induce the expression of the Red recombination system.

3. When the OD₆₀₀ value of the culture medium in step 2 reached 0.6, 5μL of the 300 ng/μL linear targeting DNA fragment 2 as prepared in step(I) was taken and used to electroporate and transform S. paratyphiCMCC50973Δcld/pKOBEG.

4. 1 mL of pre-cooled low salt LB liquid medium was rapidly added to thetransformed cells, resuscitation was performed at 30° C. for about 2.5hours, and then the medium was coated on LB plates containing kanamycinat a concentration of 50 μg/mL, placed in 30° C. incubator and culturedovernight, and positive clones were screened out.

5. The positive clones were inoculated into a liquid LB medium (withkanamycin resistance at a concentration of 50 μg/mL), cultured andpassaged twice at 42° C. (12 hours each time) to remove pKOBEG plasmid,and finally obtain a mutant with kanamycin resistance waaL deletion,named S. paratyphi CMCC50973ΔcldwaaL::kan.

(IV) The plasmid pCP20 coding FRT site-specific recombinase waselectroporated and transferred into S. paratyphi CMCC50973ΔcldwaaL::kan,cultured at 30° C. on a LB plate that contained chloramphenicol at aconcentration of 50 μg/mL and was free of kanamycin, positive clones ofCm^(r)Km^(s) (chloramphenicol resistant, Kanamycin sensitive) werescreened out.

(V) The positive clones screened out in step (IV) were transferred intoa liquid LB and cultured at 42° C. for 12 h to obtain a mutant withdeletion of target genes that did not contain kanamycin and plasmidpCP20, which were named as S. paratyphi CMCC50973ΔcldΔwaaL.

II. Molecular Identification of S. paratyphi CMCC50973ΔcldΔwaaL

The genomic DNAs of S. paratyphi CMCC509730cld and S. paratyphiCMCC50973ΔcldΔwaaL were separately used as templates, and PCRidentification was performed by separately using a pair of waaL internalprimers (73 waaLn1/73 waaLn2), a pair of waaL external primers(73waaLw1/73waaLw2) and kan primers (kan1/kan2). The results are shownin FIG. 8; and in FIG. 8, 50973ΔwaaL represents S. paratyphiCMCC50973ΔcldΔwaaL; 50973 represents S. paratyphi CMCC50973Δcld.

The results showed that there was not a target strip when the PCRamplification was performed by using the genomic DNA of S. paratyphiCMCC50973ΔcldΔwaaL as template and waaL internal primers as primers;while there was a target strip when the PCR amplification was performedby using the genomic DNA of S. paratyphi CMCC50973Δcld as template andwaaL internal primers as primers. Moreover, since the S. paratyphiCMCC50973ΔcldΔwaaL had knockout of waaL gene, the target strip obtainedwhen performing the PCR amplification by using the genomic DNA of S.paratyphi CMCC50973ΔcldΔwaaL as template and waaL external primers asprimers was smaller than the strip obtained when performing PCRamplification by using the genomic DNA of S. paratyphi CMCC50973Δcld astemplate and waaL external primers as primers. As a result of theremoval of the Kan resistance gene, there was not a target strip whenthe PCR amplification was performed by using the genomic DNA of S.paratyphi CMCC50973ΔcldΔwaaL as a template and the kan primer as aprimer.

These results demonstrated the successful construction of the Salmonellaparatyphi A 50973 mutant S. paratyphi CMCC50973ΔcldΔwaaL, which had waaLgene deletion and cld gene deletion.

III. Construction of Glycosylation Engineering Salmonella paratyphi A

1. Construction of rEPA4573 and rCTB4573 Expression Vectors

A recombinant Pseudomonas aeruginosa exotoxin A fusion protein(rEPA4573) was constructed according to the amino acid sequence ofPseudomonas aeruginosa exotoxin A (AE004091.2) published by GeneBank, inwhich its signal peptide (the first 25 amino acids) was replaced by DsbAsignal peptide, its E at position 553 was deleted, in the meantime, theL at position 552 was mutated as V, and its C-terminal was fused withthe polypeptide sequences shown in positions 45-73 amino acids (definedas Pla) of Neisseria meningitidis pilin PiLE (NC_003112.2) and 6×Histag. The amino acid sequence of the optimized rEPA4573 was shown in SEQID NO: 4, wherein the 1-19 positions were the amino acid sequence of theDsbA signal peptide; the amino acids at positions 20-631 were the aminoacid sequence of non-toxic mutant of Pseudomonas aeruginosa toxinprotein A; the amino acids 637-665 were the amino acid sequence of thepolypeptide at 45-73 positions of Neisseria meningitidis pilin PiLE(NC_003112.2), and the amino acids 666-674 were flexible linker sequenceand 6×His tag sequence; the optimized gene sequence of rEPA4573 wasshown in SEQ ID NO: 3. The artificially synthesized rEPA4573 codingsequence was digested with EcoR I and Hind III, and ligated intopMMB66EH expression vector (ATCC, ATCC37620) to constructpMMB66EH-rEPA4573 vector.

Sequencing results showed that the sequence shown in SEQ ID NO: 3 wasinserted between the EcoR I and Hind III cleavage sites of the pMMB66EHexpression vector, indicating that the vector was correct.

The recombinant CTB fusion protein (rCTB4573) was constructed accordingto the amino acid sequence of cholera toxin B subunit (CTB) (X76390.1)published by GeneBank, in which its signal peptide (the first 21 aminoacids) was replaced by the DsbA signal peptide, and the C-terminal ofthe recombinant fusion protein was fused with the polypeptide sequenceat the 45-73 positions of Neisseria meningitidis pilin PiLE(NC_003112.2) and 6×His tag. The amino acid sequence of the optimizedrecombinant CTB fusion protein was set forth in SEQ ID NO: 6, whereinthe 1-19 positions were the amino acid sequence of the DsbA signalpeptide, the positions 20-122 were the amino acid sequence of thecholera toxin B subunit, the 128-156 positions were the amino acidsequence at the 45-73 position of Neisseria meningitidis PilE(NC_003112.2), the 157-166 positions were the flexible linker and the6×His tag sequence; the coding sequence of the optimized recombinant CTBfusion protein was shown in SEQ ID NO: 5. The artificially synthesizedgene encoding recombinant CTB fusion protein was digested with EcoR Iand Hind III, and ligated into pMMB66EH expression vector (ATCC,ATCC37620) to construct pMMB66EH-rCTB4573 vector.

Sequencing results showed that the sequence shown in SEQ ID NO: 5 wasinserted between the EcoR I and Hind III restriction sites of thepMMB66EH expression vector, indicating that the vector was correct.

2. Construction of cld_(LT2) and pglL Tandem Expression Vectors

According to the amino acid sequence of the Neisseria meningitidisO-oligosaccharide transferase PglL (JN200826.1) published by GeneBank,its DNA sequence was synthesized by whole gene synthesis technique. Theamino acid sequence of Neisseria meningitidis O-oligosaccharidetransferase PglL was shown in SEQ ID NO: 8, and the gene encodingNeisseria meningitidis O-oligosaccharide transferase PglL was shown inSEQ ID NO: 7.

The artificially synthesized gene encoding PglL was digested with EcoR Iand Hind III, ligated into pKK223-3 vector (commercially available fromUppasla Pharmacia LKB Biotechniligy AB, Sweden), and primers223tac-box5′ and 223tac-box3′ were used for amplification to obtain theexpression cassette of PglL, and ligated to the Bgl II site ofpETtac28-cld_(LT2) obtained in Example 1, so as to construct thepETtac28-pglL-cld_(LT2) recombinant expression vector. The primersequences are as follows:

223tac-box5′: ATCGAGATCTACTGCATAATTCGTGTCGCTCAAG;223tac-box3′: ATCGAGATCTGTCTCATGAGCGGATACATATTTG.

3. Construction of pglL Expression Vector

The expression cassette of pglL prepared in the above step 2 was ligatedto the Bgl II site of pET28a (commercially available from Novagen) toconstruct a pETtac28-pglL recombinant expression vector.

4. Construction of S. paratyphiCMCC50973ΔcldΔwaaL/pMMB66EH-rEPA4573/pETtac28-pglL-cld_(LT2)

The pMMB66EH-rEPA4573 and pETtac28-pglL-cld_(LT2) plasmids wereelectroporated orderly into the S. paratyphi CMCC50973ΔcldΔwaaLelectroporation competent cells as prepared by the above methods, so asto construct the glycosylation engineering Salmonella paratyphi A S.paratyphi CMCC50973ΔcldΔwaaL/pMMB66EH-rEPA4573/pETtac28-pglL-cld_(LT2).

5. Construction of S. paratyphiCMCC50973ΔwaaL/pMMB66EH-rEPA4573/pETtac28-pglL

The pMMB66EH-rEPA4573 and pETtac28-pglL plasmids were electroporatedorderly into the S. paratyphi CMCC50973ΔwaaL electroporation competentcells as prepared by the above methods, so as to construct theglycosylation engineering Salmonella paratyphi A S. paratyphiCMCC50973ΔwaaL/pMMB66EH-rEPA4573/pETtac28-pglL.

6. Construction of S. paratyphiCMCC50973ΔcldΔwaaL/pMMB66EH-rCTB4573/pETtac28-pglL-cld_(LT2)

The pMMB66EH-rCTB4573 and pETtac28-pglL-cld_(LT2) plasmids wereelectroporated orderly into the S. paratyphi CMCC50973ΔcldΔwaaLelectroporation competent cells as prepared by the above methods, so asto construct the glycosylation engineering Salmonella paratyphi A S.paratyphi CMCC50973ΔcldΔwaaL/pMMB66EH-rCTB4573/pETtac28-pglL-cld_(LT2).

7. Construction of S. paratyphiCMCC50973ΔwaaL/pMMB66EH-rCTB4573/pETtac28-pglL

The pMMB66EH-rCTB4573 and pETtac28-pglL plasmids were electroporatedorderly into the S. paratyphi CMCC50973ΔwaaL electroporation competentcells as prepared by the above methods, so as to construct theglycosylation engineering Salmonella paratyphi A S. paratyphiCMCC50973ΔwaaL/pMMB66EH-rCTB4573/pETtac28-pglL.

8. Construction of S. paratyphi CMCC50973ΔwaaL/pMMB66EH-rCTB4573

The pMMB66EH-rCTB4573 plasmid was electroporated orderly into the S.paratyphi CMCC50973ΔwaaL electroporation competent cells as prepared bythe above methods, so as to construct the glycosylation engineeringSalmonella paratyphi A S. paratyphi CMCC50973ΔwaaL/pMMB66EH-rCTB4573.

9. Construction of S. paratyphi CMCC50973ΔwaaL/pMMB66EH-rEPA4573

The pMMB66EH-rEPA4573 plasmid was electroporated orderly into the S.paratyphi CMCC50973ΔwaaL electroporation competent cells as prepared bythe above methods, so as to construct the glycosylation engineeringSalmonella paratyphi A S. paratyphi CMCC50973ΔwaaL/pMMB66EH-rEPA4573.

IV. Detection of Glycosylation Situation of rEPA4573 and rCTB4573

A single clone of glycosylation engineering bacteria was picked andinoculated into a LB medium containing ampicillin at a finalconcentration of 100 μg/mL and kanamycin at a final concentration of 50μg/mL, cultured at 37° C. until OD₆₀₀ was about 0.6, and then IPTG wasadded at a final concentration of 1 mM, cooled to perform induction at16° C. for 20 h.

On the next day, 1 mL of the bacterial liquor induced at 16° C. for 20 hwas taken, centrifuged to take the bacteria, the bacteria were slowlysuspended with 1× reduction buffer, treated with boiling water bath for10 min, then subjected to SDS-PAGE electrophoresis. Afterelectrophoresis was completed, the protein was transferred to a PVDFmembrane by a Bio-Lab semi-dry transfer, the transfer was performed atconstant voltage of 20V for 1 h, and anti-His mouse monoclonal antibody(commercially available from Sigma, Cat. A7058) was used for detection,in which specific procedures could be seen in the Molecular CloningGuide.

It can be seen from FIG. 3 that the molecular weights of theglycosylation-modified rEPA4573-OPS_(Spty50973) andrCTB4573-OPS_(Spty50973) were significantly increased after the cld geneof S. paratyphi CMCC50973 itself was replaced with cld_(LT2), indicatingthat after the substitution of cld_(LT2), the polysaccharide proteinratio of glycoprotein was significantly improved.

V. Increasing the Proportion of Polysaccharides in Salmonella paratyphiA O-Polysaccharide-Recombinant CTB Fusion Protein Via TandemO-Glycosylation Sites

1. Construction of Recombinant CTB Fusion Protein (rCTB4573₃) ExpressionVector Containing Three P1a Sequences

In order to increase polysaccharide-protein ratio of glycoprotein, thepresent invention performed tandem fusion at C-terminus of CTB with 3polypeptide sequences (P1a sequence) of the positions 45-73 of Neisseriameningitidis pilin PilE (NC_003112.2), i.e., rCTB4573₃ The amino acidsequence of the recombinant CTB fusion protein was shown in SEQ ID NO:10, wherein the positions 1-19 were the amino acid sequence of the DsbAsignal peptide; the positions 20-122 were the amino acid sequence of thecholera toxin B subunit; the positions 123-127 were the flexible linker;the positions 128-222 were the amino acid sequence of the 3 polypeptidesequences of the positions 45-73 of Neisseria meningitidis pilin PilE(NC_003112.2); the positions 223-232 were 6×His tag sequence; the codingsequence of the recombinant CTB fusion protein was shown in SEQ ID NO:9. Wherein, the gene encoding the polypeptide as set forth in positions45 to 73 of Neisseria meningitidis pilin PilE (NC_003112.2) was shown inSEQ ID NO: 14, and the protein sequence encoded by this gene was shownin SEQ ID NO: 13.

The artificially synthesized coding sequence of the recombinant CTBfusion protein was digested with EcoR I and Hind III, and ligated intopMMB66EH expression vector (ATCC, ATCC37620) to constructpMMB66EH-rCTB4573₃ vector.

The sequencing results showed that the sequence shown in SEQ ID NO: 9was inserted between the EcoR I and Hind III restriction sites of thepMMB66EH expression vector, indicating that the vector was correct.

2. Construction of S. paratyphiCMCC50973ΔcldΔwaaL/pMMB66EH-rCTB4573₃/pETtac28-pglL-cld_(LT2)

The pMMB66EH-rCTB4573₃ and pETtac28-pglL-cld_(LT2) plasmids wereelectroporated into the S. paratyphi CMCC50973ΔcldΔwaaL electroporationcompetent cells as prepared by the above methods, so as to construct theglycosylation engineering S. paratyphiCMCC50973ΔcldΔwaaL/pMMB66EH-rCTB4573₃/pETtac28-pglL-cld_(LT2).

3. Preparation of rCTB4573₃-OPS_(Spty50973)

The method was the same as above mentioned.

It can be seen from FIG. 4 that after the addition of threeglycosylation sites, the recombinant CTB fusion protein underwentO-glycosylation modification, and at the same time, due to the additionof multiple glycosylation sites, the existence of multiple clusters ofglycosylation strips indicated the multiple sites were all effectiveglycosylation sites.

VI. Acquisition of rEPA4573-OPS_(Spty50973) andrCTB4573₃-OPS_(Spty50973)

1. Purification of rEPA4573-OPS_(Spty50973)

Monoclones of the glycosylation engineering strain S. paratyphiCMCC50973ΔcldΔwaaL/pMMB66EH-rEPA4573/pETtac28-pglL-cld_(LT2) were pickedout, inoculated to a LB culture plate containing ampicillin andkanamycin double resistances, cultured at 37° C. until OD₆₀₀ reachedabout 0.6, then IPTG at a final concentration of 1 mM was added, andcooled to perform induction at 25° C. for 20 h.

1) Pre-Treatment of Samples

10 g of the above bacteria after induction at 25° C. for 20 h was taken,added with 100 mL of purified water, subjected to ultrasonication(ultrasonication for 3 s and suspension for 5 s, cumulativeultrasonication time 30 min), centrifuged at 12000 g of centrifugalforce, and the supernatant was collected. To this crude extract, pH 7.5Tris-HCL at a final concentration of 20 mM, NaCL at a finalconcentration of 0.2M and imidazole at a final concentration of 10 mMwere added, fully stirred, then centrifuged again at 12000 g ofcentrifugal force, and the supernatant was collected and used as a crudeextract containing the recombinant EPA fusion protein(rEPA4573-OPS_(Spty50973)) that was modified by Salmonella paratyphi AOPS.

2) Purifying Samples with Chelating Affinity Chromatographic Column

Chelating affinity chromatographic column (Φ1.6 cm*15 cm) was used forprimary purification of samples. The column bed was first rinsed with 3column bed volumes of 0.5M NaOH, then equilibrated with deionized waterto neutral pH, then equilibrated with 3 column bed volumes of 0.5MNiSO₄, and then equilibrated with 1 column bed volume of B1 solution (20mM pH 7.5 Tris-HCl, 0.5M NaCl, 500 mM imidazole), and finallyequilibrated with 3 column bed volumes of A1 solution (20 mM pH 7.5Tris-HCl, 0.5M NaCl, 10 mM imidazole), wherein the above-used flow ratewas always 4 mL/min. The above crude extract containingrEPA4573-OPS_(Spty50096) was loaded from A tube, and the unbound proteinwas washed off with solution A, and the elution was finally performed byusing 100% B1 to collect 30 mL of eluate.

3) Desalting Samples

The sample that was preliminarily purified by the Chelating affinitychromatographic column was desalted by using G25 fine chromatographiccolumn (Φ1.6 cm*30 cm), in which the mobile phase was A3 solution (20 mMpH 7.5 Tris-HCl). The column bed was firstly rinsed with 3 column bedvolumes of 0.5M NaOH, then equilibrated with deionized water to pHneutral, and finally equilibrated with 3 column volumes of A3 solution.The sample was loaded from A tube, 60 mL of sample was collected, andthe above-used flow rate was always 4 mL/min.

4) Further Purification of rEPA4573-OPS_(Spty50973) by Using ProteinPakDEAE8HR Anion Exchange Chromatographic Column

The desalted sample was further purified by ProteinPak DEAE8HR anionexchange chromatographic column (waters). The column bed was firstrinsed with 3 column bed volumes of 0.5M NaOH, then equilibrated withdeionized water to pH neutral, and then equilibrated with 3 column bedvolumes of A3 solution (20 mM pH 7.5 Tris-HCl). The sample was loadedfrom A tube, the unbound glycoprotein was washed off with A3 solution,then linear elution was performed using 0-50% B3 solution (20 mM pH 7.5Tris-HCl, 1M NaCl) for 30 min, and the eluate was collected, in whichthe above used flow rate was always 1 mL/min. The peak position ofglycoprotein rEPA4573-OPS_(Spty50973) was at position of about 8-18mS/cm.

5) Fine Purification of rEPA4573-OPS_(Spty50973) by Using Superdex 75Chromatographic Column

The sample as purified by ProteinPak DEAE8HR anion exchangechromatographic column was further purified by using Superdex 75 FPLC(Φ1:01 cm*30 cm, GE). The column bed was first washed with 3 column bedvolumes of 0.5M NaOH, then equilibrated with deionized water to pHneutral, and then equilibrated with 3 column bed volumes of A4 solution(20 mM pH 7.5 PB, 0.9% NaCl). The sample in volume of 1 mL was loadedfrom a sample loop, and 8 to 11 mL of the effluent sample was collected,and this sample was the purified rEPA4573-OPS_(Spty50973).

This sample was analyzed by 8% SDS-PAGE and western blot, and theresults were shown in FIG. 5.

2. Purification of rCTB4573₃-OPS_(Spty50973)

Monoclone of the glycosylation engineering strain S. paratyphiCMCC50973ΔcldΔwaaL/pMMB66EH-rCTB4573₃/pETtac28-pglL-cld_(LT2) was pickedout, inoculated to a LB culture medium with ampicillin and kanamycindouble resistance, cultured at 37° C. until OD₆₀₀ was about 0.6, thenIPTG at a final concentration of 1 mM was added, and cooled to performinduction at 16° C. for 20 h.

1) Pre-Treatment of Samples

10 g of the above bacteria after induction at 16° C. for 20 h was taken,added with 100 mL of purified water, subjected to ultrasonication(ultrasonication for 3 s and suspension for 5 s, cumulativeultrasonication time 30 min), centrifuged at 12000 g of centrifugalforce, and the supernatant was collected. To this crude extract, pH 7.5Tris-HCL at a final concentration of 20 mM, NaCL at a finalconcentration of 0.2M and imidazole at a final concentration of 10 mMwere added, fully stirred, then centrifuged again at 12000 g ofcentrifugal force, and the supernatant was collected and used as a crudeextract containing the recombinant CTB fusion protein(rCTB4573₃-OPS_(Spty50973)) that was modified by Salmonella paratyphi AO-polysaccharide.

2) Purifying Samples with Chelating Affinity Chromatographic Column

Chelating affinity chromatographic column (Φ1.6 cm*15 cm) was used forprimary purification of samples. The column bed was first rinsed with 3column bed volumes of 0.5M NaOH, then equilibrated with deionized waterto neutral pH, then equilibrated with 3 column bed volumes of 0.5MNiSO₄, and then equilibrated with 1 column bed volume of B1 solution (20mM pH 7.5 Tris-HCl, 0.5M NaCl, 500 mM imidazole), and finallyequilibrated with 3 column bed volumes of A1 solution (20 mM pH 7.5Tris-HCl, 0.5M NaCl, 10 mM imidazole), wherein the above-used flow ratewas always 4 mL/min.

The above crude extract containing rCTB4573₃-OPS_(Spty50973) was loadedfrom A tube, and the unbound protein was washed off with solution A, andthe elution was finally performed by using 100% B1 to collect 30 mL ofeluate.

3) Desalting Samples

The sample that was preliminarily purified by the Chelating affinitychromatographic column was desalted by using G25 fine chromatographiccolumn (Φ1.6 cm*30 cm), in which the mobile phase was A2 solution (20 mMpH 5.4HAc—NaAc). The column bed was firstly rinsed with 3 column bedvolumes of 0.5M NaOH, then equilibrated with deionized water to pHneutral, and finally equilibrated with 3 column volumes of A2 solution.The sample was loaded from A tube, 60 mL of sample was collected, andthe above-used flow rate was always 4 mL/min.

4) Further Purification of rCTB4573₃-OPS_(Spty50973) by Using ProteinPakSP8HR Cation Exchange Chromatographic Column

The desalted sample was further purified by ProteinPak SP8HR cationexchange chromatographic column (waters). The column bed was firstrinsed with 3 column bed volumes of 0.5M NaOH, then equilibrated withdeionized water to pH neutral, and then equilibrated with 3 column bedvolumes of A2 solution (20 mM pH5.4 HAc—NaAc). The sample was loadedfrom A tube, the unbound glycoprotein was washed off with A2 solution,then linear elution was performed using 0-50% B2 solution (20 mM pH5.4HAc—NaAc, 1M NaCl) for 30 min, and the eluate was collected, in whichthe above used flow rate was always 1 mL/min. The peak position ofglycoprotein rCTB4573₃-OPS_(Sp50973) was at position of about 35-45mS/cm. The sample was analyzed by 12% SDS-PAGE and western blot, and theresults were shown in FIG. 6.

VII. Preparation and Animal Experimental Evaluation ofPolysaccharide-Protein Conjugate Vaccines of rCTB4573₃-OPS_(Spty50973)and rEPA4573-OPS_(Spty50973)

1. Preparation of Polysaccharide-Protein Conjugate Vaccines ofrCTB4573₃-OPS_(Spty50973) and rEPA4573-OPS_(Spty50973)

The purified rCTB4573₃-OPS_(Spty50973) and rEPA4573-OPS_(Spty50973) weresterilized by filtration, and mixed with aluminum hydroxide adjuvant(Rehydragel LV, General Chemical) at a ratio of 9:1.

2. Preparation of O-Antigen (OPS_(Spty50973))

LPS was firstly extracted by hot phenol-water method (SUN Yang, FENGShuzhang, ZHU Lingwei, et al, “Preparation and identification ofEnterohemorrhagic Escherichia coli O157 LPS monoclonal antibody, [J].Journal of Zoonoses of China, 2007, 23 (10): 971-973), preserved bylyophilization, then dissolved with 1% glacial acetic acid at aconcentration of 10 mg/ml, treated with boiling water bath for 90minutes, then cooled to room temperature, and adjusted to pH 7.0. Thesupernatant was collected after centrifugation at 64,000×g for 5 hours,and thoroughly dialyzed with deionized water and preserved bylyophilization.

3. Animal Immunization and Effect Evaluation of Conjugate Vaccines ofrCTB4573₃-OPS_(Spty50973) and rEPA4573-OPS_(Spty50973)

40 female Balb/c mice of 6 weeks old were randomly divided into 4groups. Aluminum hydroxide, OPS_(Spty50973), rCTB4573₃-OPS_(Spty50973)and rEPA4573-OPS_(Spty50973) samples were separately injected intomuscles of the 4 group of mice, in which the aluminum hydroxide groupwas negative control, the other three groups were injected with 10 μgpolysaccharide in an amount expressed in polysaccharide content; andblood samples were taken separately on the 1^(st), 22^(nd) and 50^(th)day after immunization, and on the 10^(th) day after the thirdimmunization.

The antibody titer of anti-Salmonella paratyphi A O-polysaccharide inmice serum of each group was measured by indirect ELISA method. Theenzyme-linked plate was coated with the extracted Salmonella paratyphi ALPS, in which each well was coated with 10 μg of LPS, and otherprocedures could be seen in “A Guide to Experimental BiomedicalBiology”.

It can be seen from FIG. 7, both of the rCTB4573₃-OPS_(Spty50973) andrEPA4573-OPS_(Spty50973) as prepared in the present invention viaextending OPS of Salmonella paratyphi A CMCC50973 can induce mice toproduce specific antibodies against Salmonella paratyphi A CMCC50973OPS, in which the antibody titer increased significantly in comparisonwith the OPS group with injection of OPS only.

INDUSTRIAL APPLICATIONS

In comparison with Paratyphoid A polysaccharide-protein conjugatevaccines in the prior art, the present invention prepares Paratyphoid Apolysaccharide-protein conjugate vaccines by using an O-antigen withextended carbohydrate chain by one-step bio-crosslinking method. Themethod comprises: using Salmonella paratyphi A as a host strain whereinthe O-antigen chain length was extended and O-antigen ligase gene waaLwas deleted, co-expressing a recombinant fusion protein gene and aNeisseria meningitidis O-oligosaccharide transferase gene pglL in thehost strain, using the O-antigen of the host strain Salmonella paratyphiA to directly modify the recombinant fusion protein in manner ofO-glycosylation modification, so as to obtain O-antigen-modifiedrecombinant fusion protein of Salmonella paratyphi A and to prepareParatyphoid A polysaccharide-protein conjugate vaccinesrCTB4573₃-OPS_(Spty50973) and rEPA4573-OPS_(Spty50973). The resultsshowed that the vaccines prepared by the present invention can induce inmice the generation of specific antibody against Salmonella paratyphi ACMCC50973 OPS, and the antibody titer is obviously improved incomparison with the mice with injection of OPS alone.

Unless otherwise indicated, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention pertains. Exemplary methods andmaterials are described below. Although methods and materials similar orequivalent to those described in the text can also be used to carry outthe present invention, they are obvious for those skilled in the art.All publications and other references mentioned herein are incorporatedby reference in their entirety. In the event of inconsistencies, thepresent specification including definitions should prevail. Allmaterials, methods and examples above mentioned are illustrative onlyand not restrictive.

1. A recombinant strain, which is obtained by introducing a cld_(LT2)gene encoding an enzyme controlling chain length of O-antigen ofSalmonella typhimurium into Salmonella paratyphi A which is deficient incld gene encoding an enzyme controlling chain length of O-antigen. 2.The recombinant strain according to claim 1, characterized in that theenzyme controlling chain length of O-antigen of Salmonella typhimuriumhas an amino acid sequence with at least 90% identity to the amino acidsequence as shown in SEQ ID NO:
 2. 3. The recombinant strain accordingto claim 1, characterized in that the Salmonella paratyphi A which isdeficient in cld gene encoding an enzyme controlling chain length ofO-antigen is obtained by a method comprising the following steps: (1)preparing a linear targeting DNA fragment 1, which has a nucleotidesequence as shown in SEQ ID NO: 11, and contains a cat gene; (2)transforming pKD46 plasmid into a Salmonella paratyphi strain, to obtaina recombinant strain designated as S. paratyphi/pKD46; (3) inducingexpression of Red recombination system in the S. paratyphi/pKD46 strain,and transforming the linear targeting DNA fragment 1 into the S.paratyphi/pKD46 strain, so that the linear targeting DNA fragment 1replaces the cld gene of the S. paratyphi/pKD46 strain to obtain arecombinant strain designated as S. paratyphi cld::cat/pKD46; (4)deleting the pKD46 plasmid from the S. paratyphi cld::cat/pKD46 strain,to obtain a recombinant strain designated as S. paratyphi cld::cat; theS. paratyphi cld::cat is a S. paratyphi in which cld gene sequence issubstituted with cat gene sequence; (5) transforming plasmid pCP20 intothe S. paratyphi cld::cat and deleting cat gene, to obtain a recombinantstrain designated as S. paratyphi Δcld; the S. paratyphi Δcld is a cldgene-deleted S. paratyphi.
 4. A method for extending carbohydrate chainlength of O-antigen of Salmonella paratyphi A, comprising the followingsteps: culturing the recombinant strain according to claim 1 to expressthe cld_(LT2) gene, so that the recombinant strain synthesizes anO-antigen of which carbohydrate chain length is extended.
 5. AnO-antigen, which is prepared by the method according to claim
 4. 6.(canceled)
 7. An method for preparing a vaccine for prevention and/ortreatment of a disease caused by Salmonella paratyphi A by one-stepbio-crosslinking, comprising the steps of: (1) inactivating cld geneencoding an enzyme controlling chain length of O-antigen and waaL geneencoding O-antigen ligase of a Salmonella paratyphi A strain, to obtaina Salmonella paratyphi A with double deletion of cld gene and waaL gene;(2) introducing cld_(LT2) gene encoding an enzyme controlling chainlength of O-antigen of Salmonella typhimurium, pglL gene encodingO-oligosaccharyltransferase of Neisseria meningitidis and a gene codingrecombinant fusion protein into the Salmonella paratyphi A with doubledeletion of cld gene and waaL gene, to obtain a recombinant strain; (3)culturing the recombinant strain to obtain recombinant fusion proteinwith O-antigen-modified, and processing the recombinant fusion proteinwith O-antigen-modified to obtain the desired vaccine.
 8. The methodaccording to claim 7, characterized in that, the Salmonella paratyphi Awith double deletion of cld gene and waaL gene is constructed by amethod comprising the following steps: (1) preparing a linear targetingDNA fragment 2, which has a nucleotide sequence as shown in SEQ ID NO:12, and which contains a kan gene; (2) transforming pKOBEG plasmid intothe Salmonella. ParatyphiΔcld strain as defined in claim 3, to obtain arecombinant strain designated as S. paratyphiΔcld/pKOBEG; (3) inducingexpression of Red recombination system in the S. paratyphiΔcld/pKOBEGstrain, and transforming the linear targeting DNA fragment 2 into the S.paratyphiΔcld/pKOBEG strain, so that the linear targeting DNA fragment 2replaces the waaL gene of the S. paratyphiΔcld/pKOBEG strain to obtain arecombinant strain designated as S. paratyphiΔcld waal::kan/pKOBEG; (4)deleting the pKOBEG plasmid from the S. paratyphiΔcld waal::kan/pKOBEGstrain, to obtain a recombinant strain designated as S.paratyphiΔcldwaaL::kan; the S. paratyphiΔcldwaaL::kan is a S.paratyphiΔcld in which waaL gene sequence is substituted with kan genesequence; (5) transforming plasmid pCP20 into the S.paratyphiΔcldwaaL::kan and deleting kan gene, to obtain a recombinantstrain designated as S. paratyphiΔcldΔwaaL; the S. paratyphiΔcldΔwaaL isa S. paratyphi with deletion of cld gene and waaL gene.
 9. The methodaccording to claim 7, characterized in that, the recombinant fusionprotein comprises a N-terminal signal peptide, a carrier protein, and apeptide fragment comprising a serine as an O-glycosylation site atposition 63 of Neisseria meningitidis pilin PilE; the N-terminal signalpeptide is PelB signal peptide, DsbA signal peptide, STII signalpeptide, OmpA signal peptide, PhoA signal peptide, LamB signal peptide,SpA signal peptide or Enx signal peptide; the carrier protein is anon-toxic mutant of a bacterial toxin protein or a fragment of abacterial toxin protein; the peptide fragment comprising a serine as anO-glycosylation site at position 63 of Neisseria meningitidis pilin PilEis a peptide fragment with an amino acids as set forth in positions45-73 of Neisseria meningitidis pilin PilE.
 10. The method according toclaim 9, characterized in that, the bacterial toxin protein isPseudomonas aeruginosa exotoxin A, cholera toxin, diphtheria toxin ortetanus toxin.
 11. The method according to claim 9, characterized inthat, the non-toxic mutant of the bacterial toxin protein is a non-toxicmutant of Pseudomonas aeruginosa exotoxin A or a non-toxic mutant ofdiphtheria toxin; the fragment of the bacterial toxin protein is a Bsubunit of cholera toxin or a C protein of tetanus toxin.
 12. The methodaccording to claim 9, characterized in that, the N-terminal signalpeptide is a DsbA signal peptide.
 13. The method according to claim 7,characterized in that, the cld_(LT2) gene encoding an enzyme controllingchain length of O-antigen of Salmonella typhimurium and the pglL geneencoding O-oligosaccharide transferase of Neisseria meningitidis areintroduced into the Salmonella paratyphi A with double deletion of cldgene and waaL gene through pETtac28-pglL-cld_(LT2) recombinantexpression vector; the gene coding the recombinant fusion protein isintroduced into the Salmonella paratyphi A with double deletion of cldgene and waaL gene through a pMMB66EH-rCTB4573 recombinant expressionvector or a pMMB66EH-rEPA4573 recombinant expression vector or apMMB66EH-rCTB4573₃ recombinant expression vector; thepETtac28-pglL-cld_(LT2) recombinant expression vector is constructed bya method comprising: the cld_(LT2) gene encoding an enzyme controllingchain length of O-antigen of Salmonella typhimurium and the pMMB66EHvector are digested by restriction endonucleases EcoR I and Hind III,and are ligated to obtain a transition vector pMMB66EH-cld_(LT2);transition vector pMMB66EH-cld_(LT2) is used as template, and anexpression cassette of cld_(LT2) is obtained by amplification; theexpression cassette of cld_(LT2) and pET28a vector are digested withrestriction endonuclease Xba I and Xho I, and are ligated to obtainpETtac28-cld_(LT2) vector; pglL gene encoding O-oligosaccharidetransferase of Neisseria meningitidis and pKK223-3 vector are digestedby restriction endonucleases EcoR I and Hind III, and are ligated toobtain pKK223-3-pglL vector; pKK223-3-pglL vector is used as template,and an expression cassette of PglL is obtained by amplification; theexpression cassette of PglL and the pETtac28-cld_(LT2) vector aredigested by restriction endonuclease Bgl II, and are ligated to obtainthe pETtac28-pglL-cld_(LT2) recombinant expression vector; thepMMB66EH-rEPA4573 recombinant expression vector is constructed by amethod comprising: ligating the gene encoding fusion protein rEPA4573 ofrecombinant Pseudomonas aeruginosa exotoxin A into a multiple cloningsite of pMMB66EH vector to obtain the pMMB66EH-rEPA4573 recombinantexpression vector; the pMMB66EH-rCTB4573 recombinant expression vectoris constructed by a method comprising: the gene encoding fusion proteinrCTB4573 of recombinant cholera toxin B subunit is ligated into amultiple cloning site of pMMB66EH vector to obtain the pMMB66EH-rCTB4573recombinant expression vector; the pMMB66EH-rCTB4573₃ recombinantexpression vector is constructed by a method comprising: the geneencoding fusion protein rCTB4573₃ of recombinant cholera toxin B subunitis ligated into a multiple cloning site of pMMB66EH vector to obtain thepMMB66EH-rCTB4573₃ recombinant expression vector.
 14. The methodaccording to claim 13, characterized in that: the enzyme controllingchain length of O-antigen of Salmonella typhimurium has an amino acidsequence as shown in SEQ ID NO: 2; the Neisseria meningitidisoligosaccharyltransferase pglL has an amino acid sequence as shown inSEQ ID NO: 8; the fusion protein rEPA4573 of recombinant Pseudomonasaeruginosa exotoxin A has an amino acid sequence as shown in SEQ ID NO:4; the fusion protein rCTB4573 of recombinant cholera toxin B subunithas an amino acid sequence as shown in SEQ ID NO: 6; the fusion proteinrCTB4573₃ of recombinant cholera toxin B subunit has an amino acidsequence as shown in SEQ ID NO: 10; the peptide with amino acid sequenceas set forth in position 45-73 of Neisseria meningitidis pilin PilE hasan amino acid sequence as shown in SEQ ID NO:
 14. 15. A vaccine asprepared by the method according to claim
 7. 16. A method forprophylaxis and/or treatment of a disease caused by Salmonella paratyphiA, comprising a step of administering, to a subject in need, aneffective amount of the vaccine according to claim
 15. 17. Therecombinant strain according to claim 1, wherein the enzyme controllingchain length of the O-antigen of Salmonella typhimurium has an aminoacid sequence as shown in SEQ ID NO:
 2. 18. The recombinant strainaccording to claim 1, wherein the gene encoding the enzyme controllingchain length of the O-antigen of Salmonella typhimurium has a sequenceas shown in SEQ ID NO: 1.